SPINK6 inhibits human airway serine proteases and restricts influenza virus activation

Abstract SPINK6 was identified in human skin as a cellular inhibitor of serine proteases of the KLK family. Airway serine proteases are required to cleave hemagglutinin (HA) of influenza A viruses (IAVs) to initiate an infection in the human airway. We hypothesized that SPINK6 may inhibit common airway serine proteases and restrict IAV activation. We demonstrate that SPINK6 specifically suppresses the proteolytic activity of HAT and KLK5, HAT‐ and KLK5‐mediated HA cleavage, and restricts virus maturation and replication. SPINK6 constrains the activation of progeny virions and impairs viral growth; and vice versa, blocking endogenous SPINK6 enhances HA cleavage and viral growth in physiological‐relevant human airway organoids where SPINK6 is intrinsically expressed. In IAV‐infected mice, SPINK6 significantly suppresses viral growth and improves mouse survival. Notably, individuals carrying the higher SPINK6 expression allele were protected from human H7N9 infection. Collectively, SPINK6 is a novel host inhibitor of serine proteases in the human airway and restricts IAV activation.

1. The initial findings were based on human polymorphisms in the SPINK6 gene. While these were interesting data to present, experiments performed subsequently did not consider the impact of these polymorphisms on SPINK6 function. Rather, the mutated SPINK6 used in this study contained a loss-of-function R19A mutation. If this change correlates with the rs1432689 SNP, the authors should indicate this more clearly. I do not have extensive experience in human genetics, so please let me know if there is something that I am missing here. I basically want to determine if the human connection between the GWAS study and the experiments that were subsequently performed are related.
2. In the flow cytometry data presented in Figure 5, would it be possible for the authors to provide the individual histograms for the fluorescent channels as well as the plots showing the co-staining in 4 quadrants. It was difficult to see where the populations were in the plots presented, making it unclear how it was determined that 30% of the cells in the organoids expressed SPINK6. 3. The cell culture studies presented variable MOIs for inoculation, making it difficult to compare some of the infections across the full manuscript. 4. In the murine study, would the authors be willing to describe how they decided the dose and delivery for SPINK6. While the survival data were interesting, and supported the hypothesis, the weight loss and lung titer data did not match well with survival results. Specifically, with the large error bars in the weight loss data, I wonder if there were two different responses within the challenge groups with some mice losing more weight than others. If this is the case, the authors should clearly present these differences in response to the virus infection. 5. Further, the virus titers were quite high in both groups, indicating that SPINK6 does not effectively eliminate the virus infection. Would a different dose of SPINK6 have a greater effect on virus infectivity? 6. Overall, as presented, it is not clear that the conclusions from the results presented fully support the title which implies that SPINK6 has a much greater effect on virus propagation in living systems, especially in humans.

Referee #1 (Remarks for Author):
Comments for the authors of the EMBO Molecular Medicine manuscript number EMM-2021-13691: The authors of the EMBO Molecular Medicine manuscript "SPINK6 inhibits human airway serine proteases and restricts influenza virus activation", present their findings related to the impact of proteases and anti-proteases on influenza hemagglutinin cleavage and infection. Specifically, they focus on the serine protease inhibitor Kazal-type 6 (SPINK6), human data that correlates genetic variation in SPINK 6 with susceptibility to H7N9 infection. The authors then proceed to evaluate HA cleavage from HA0 into HA2 in the presence of known proteases, using SPINK6 as an anti-protease. They draw the conclusion that SPINK6 inhibits HAT and KLK5 protease activity, which restricts HA cleavage and is associated with reduced infectivity in human organoid and murine infection models. Their overall conclusion is that SPINK6 is an inhibitor of serine proteases in a manner that restricts influenza A virus infection in the human respiratory tract. While these findings interesting, and the experimental progression both logical and well-designed, I have identified come limitations to the study that will be presented below. General Comments: 1. The initial findings were based on human polymorphisms in the SPINK6 gene. While these were interesting data to present, experiments performed subsequently did not consider the impact of these polymorphisms on SPINK6 function. Rather, the mutated SPINK6 used in this study contained a loss-of-function R19A mutation. If this change correlates with the rs1432689 SNP, the authors should indicate this more clearly. I do not have extensive experience in human genetics, so please let me know if there is something that I am missing here. I basically want to determine if the human connection between the GWAS study and the experiments that were subsequently performed are related. 2. In the flow cytometry data presented in Figure 5, would it be possible for the authors to provide the individual histograms for the fluorescent channels as well as the plots showing the co-staining in 4 quadrants. It was difficult to see where the populations were in the plots presented, making it unclear how it was determined that 30% of the cells in the organoids expressed SPINK6.
3. The cell culture studies presented variable MOIs for inoculation, making it difficult to compare some of the infections across the full manuscript. 4. In the murine study, would the authors be willing to describe how they decided the dose and delivery for SPINK6. While the survival data were interesting, and supported the hypothesis, the weight loss and lung titer data did not match well with survival results. Specifically, with the large error bars in the weight loss data, I wonder if there were two different responses within the challenge groups with some mice losing more weight than others. If this is the case, the authors should clearly present these differences in response to the virus infection. 5. Further, the virus titers were quite high in both groups, indicating that SPINK6 does not effectively eliminate the virus infection. Would a different dose of SPINK6 have a greater effect on virus infectivity? 6. Overall, as presented, it is not clear that the conclusions from the results presented fully support the title which implies that SPINK6 has a much greater effect on virus propagation in living systems, especially in humans. Specific comments 1. There were numerous times in the reading of the manuscript where the sentence structure and grammar could be improved. 2. In Figure 2D, it was not entirely clear that the wtSPINK6 was provided as a recombinant protein. Please make this clear within the figure. 3. Large portions of the Discussion section simply repeated the results rather than presenting them in the context of the field.
Referee #2 (Remarks for Author): The authors have identified in this study a serine protease inhibitor, SPNK6, that interferes with proteolytic activation of influenza A viruses of subtypes H7 and H1. They show that SPNIK6 inhibits trypsin-mediated cleavage of virus replication in vivo and blocks activation by the airway-specific proteases HAT and KLK5. Interestingly, it does not inhibit TMPRSS2 and matriptase, other serine proteases also known to activate H7 and H1 influenza viruses. Furthermore, it is shown that SPINK6 is expressed intrinsically in human airway organoids where it also interferes with virus replication. Similar observations have been made in a mouse model. These are very interesting observations. It has long been known that ptoteilytic activation which is an important determinant of tissue tropism, host range and pathogenicity of influenza and many other viruses depends on the structure of the viral substrate, in this case the hemagglutinin, and the substrate specificity of the host protease. From the present study it is now clear that the specificity of proteolytic activation depends also on a third group of factors, protease inhibitors provided by the host.

Points of objection.
Lines 109-111: The authors refer here to a study by Chen et al., 2015, which shows that galectin regulates susceptibility to H7N9 infection. According to the authors, this observation suggests an involvement of SPINK6 in protection from virus infection. However the link between galectin and SPINK6 remains unclear. This link has to be explained.. 1. Very low animal numbers are used not allowing any significant conclusion. 2. Virus infectious dose of 5 pfu is far too low for reproducible infection. The results can be simply due to the variations in viral inoculation efficiency. 3. The animal protocol is not well described (e.g. humane endpoints are not defined).
Referee #3 (Remarks for Author): The study by Wang et al. describes a previously reported inhibitor of serine protease families, namely SPINK6. The authors show that SPINK6 inhibits proteolytic cleavage of influenza HA and inhibits viral gene expression. The authors further explored the therapeutic potential of SPINK6 in mice. While some observations are interesting, the study is at a very premature stage. Following major concerns dampen my enthusiasm: 1. Database analysis reveal according to the authors a potential role for SPINK6 SNPs in human H7N9 infection by increased transcription. This is a potential important findings that needs to be experimentally validated. Data shown in Figure 1 are in silico data. The authors should use human biopsy material and assess SPINK6 expression in relation to key proteases, such as HAT or TMPRSS2 in the upper and lower respiratory tract. Also, they should study whether viral infection affects SPINK6 mRNA expression that could have major implications on the infection course.
2. There are no data showing that SPINK6 actually inhibits H7N9 replication. In Figure  2E only vRNA amounts are shown. The authors should show infectious virus titres as p.f.u. over several time points post infection.
3. Animal data shown in Figure 6 are not allowing any conclusions. The authors have used a virus infectious dose of 5 p.f.u., which is too low to allow reproducible animal infection yet any meaningful conclusions. It is not clear why the authors used H1N1 influenza instead of H7N9 that is the primary focus of the study. It was repeatedly shown that H7N9 is able to infect mice and cause weight loss. The authors have used only 4 mice per time point assessed in Figure 6c. This is statistically not sufficient to allow significant conclusions. Further, the animal protocol is poorly described in the M&M section. There is no description of the narcosis used for infection and treatment. Humane endpoints are not defined.
1. The initial findings were based on human polymorphisms in the SPINK6 gene. While these were interesting data to present, experiments performed subsequently did not consider the impact of these polymorphisms on SPINK6 function. Rather, the mutated SPINK6 used in this study contained a loss-of-function R19A mutation. If this change correlates with the rs1432689 SNP, the authors should indicate this more clearly. I do not have extensive experience in human genetics, so please let me know if there is something that I am missing here. I basically want to determine if the human connection between the GWAS study and the experiments that were subsequently performed are related.
A: We appreciate the reviewer's comment. The integrative analysis of genetic association and eQTL suggested that higher SPINK6 expression may confer protection from human H7N9 infection. In combination with other biological evidence described in the introduction section, we formulated the hypothesis that SPINK6 may inhibit common HA cleavage serine proteases, besides the previously characterized targets KLK5 and KLK12. In the following experiments, we've used the conventional gain-of-function and/or loss-of-function experiments to demonstrate the role of SPINK6 for HA cleavage and IAV replication mediated by serine proteases. The mutant SPINK6 with a defective protease inhibition domain has nothing to with SNP rs1432689. The association SNP rs1432689 is not involved in protein encoding, instead it is correlated to the differential mRNA expression levels of SPINK6 ( Figure 6). The possible genetic basis for the correlation has been described in the text (page 10 line 280 in the current manuscript).
Thanks to the reviewer's comment, we realize that data presentation in the original manuscript was inappropriate so that the reviewer may be confused. In fact, the other biological evidence we have gleaned from previous studies (mentioned in the introduction section) is sufficient to formulate the hypothesis, without the results of the human genetic association studies. Therefore, we re-organized the data presentation. In the revised manuscript, the genetic association and eQTL are presented at the end of the results as a supportive evidence in humans. This is actually the most common way of data presentation in many similar studies.
2. In the flow cytometry data presented in Figure 5, would it be possible for the authors to provide the individual histograms for the fluorescent channels as well as the plots showing the co-staining in 4 quadrants. It was difficult to see where the populations were in the plots presented, making it unclear how it was determined that 30% of the cells in the organoids expressed SPINK6.
A: We amended the figure of flow cytometry results ( Figure 4A) as suggested by the reviewer. The y axis and x axis represent the expression of HAT and SPINK6 respectively. The quadrants are set to delineate HAT+, SPINK6+ and the negative populations, the number in each quadrant represents the percentage of cells within the quadrant. Hopefully, we have addressed the reviewer's inquiry.
A: We have designed various experiments to demonstrate the role of SPINK6 for virus replication driven by exogenous proteases (overexpression of selected proteases or the addition of trypsin in culture media) or endogenous proteases (in airway organoids). In these experiments, we have to optimize various elements, which are very distinct in different experimental settings. Overall, we aimed to achieve an active viral growth under various settings, then examined the effect of SPINK6 on viral growth. For example, in infection experiments in cell lines, the addition of TPCK-trypsin ( Figure 1E) enabled HA activation with a higher potency/efficiency than overexpression of selected proteases ( Figure 3A), which is well expected. Thus, a lower MOI inoculation was done in the former than the latter. Despite distinct MOIs in different experimental settings, we believe the same conclusion has been reached, i.e., SPINK6 inhibits HAT-and KLK5-mediated HA cleavage and viral growth.
4. In the murine study, would the authors be willing to describe how they decided the dose and delivery for SPINK6. While the survival data were interesting, and supported the hypothesis, the weight loss and lung titer data did not match well with survival results. Specifically, with the large error bars in the weight loss data, I wonder if there were two different responses within the challenge groups with some mice losing more weight than others. If this is the case, the authors should clearly present these differences in response to the virus infection.
A: We appreciate the reviewer's comments. Firstly, we chose to deliver SPINK6 protein by intranasal administration since it is a more effective delivery route to reach the infection site (mouse lung) than other routes such as intra-peritoneal injection or intra-venous injection. In addition, due to the challenge of respiratory delivery, we performed pilot experiments with various doses of protein solutions. Based on the results, we elected an experimental scheme of multiple dosing (3 times) with 10ug protein in a volume of 20ul. Under this setting, two groups of mice appeared tolerated to the administration and displayed indistinguishable response right after the manipulation. Moreover, we have demonstrated in the in vitro experiments that the addition of SPINK6 protein onto 2D airway organoids can inhibit protease activity, HA cleavage and viral growth ( Figure 4). Hence, the intranasal administration of SPINK6 was performed as an equivalent manipulation in mice. We have revised the manuscript accordingly on page 9.
We agree with the reviewer on the big error bar in body weight data. We believe it is related to the experimental design. Apart from the virus inoculation, we performed intranasal delivery of wildtype and mutant SPINK6 proteins for 3 times. The multiple dosing exerted additional stress on these virus-infected mice and exacerbated the infection in both groups, some of which may lose more body weight than the others, leading to a big variation in the body weight. In the mutant SPINK6 treatment group, the mice developed more severe disease and lost more body weight with 7 deaths on day 4 ~ day 6, only 3 mice survived the infection. Accordingly, the variation of body weight was even bigger on day 3 ~ day 5 and it became smaller from day 6 when the heavily-sick mice had died. We have described different manifestations of two groups of mice on page 9 in the revised manuscript, as suggested by the reviewer.
5. Further, the virus titers were quite high in both groups, indicating that SPINK6 does not 3 effectively eliminate the virus infection. Would a different dose of SPINK6 have a greater effect on virus infectivity? A: We agree with the reviewer that SPINK6 did not eliminate the infection. we'd like to emphasize that, in this experiment, 3-time intranasal administration of protein solutions itself was quite harsh, and aggravated the viral infection. For mice of 6~8 week-old, we have used the maximal dose and volume tolerable to the mice based on our pilot experiments. The aim of the mouse experiment is to verify the effect of SPINK6 demonstrated in vitro. Despite the failure to eliminate virus infection by SPINK6 treatment, we may have adequately fulfilled the aim. A more effective and less invasive approach of delivery is definitely required for developing SPINK6 as an effective therapeutics against influenza.
6. Overall, as presented, it is not clear that the conclusions from the results presented fully support the title which implies that SPINK6 has a much greater effect on virus propagation in living systems, especially in humans.
A: We respectfully disagree with the comment. Probably our suboptimal data presentation in the previous manuscript was unable convince the reviewer. We hope the reviewer would appreciate the revised manuscript.
Referee #2 (Remarks for Author): The authors have identified in this study a serine protease inhibitor, SPNK6, that interferes with proteolytic activation of influenza A viruses of subtypes H7 and H1. They show that SPNIK6 inhibits trypsin-mediated cleavage of virus replication in vivo and blocks activation by the airway-specific proteases HAT and KLK5. Interestingly, it does not inhibit TMPRSS2 and matriptase, other serine proteases also known to activate H7 and H1 influenza viruses. Furthermore, it is shown that SPINK6 is expressed intrinsically in human airway organoids where it also interferes with virus replication. Similar observations have been made in a mouse model. These are very interesting observations. It has long been known that ptoteilytic activation which is an important determinant of tissue tropism, host range and pathogenicity of influenza and many other viruses depends on the structure of the viral substrate, in this case the hemagglutinin, and the substrate specificity of the host protease. From the present study it is now clear that the specificity of proteolytic activation depends also on a third group of factors, protease inhibitors provided by the host.
A: We appreciate the reviewer's insightful and encouraging comments.

Points of objection.
Lines 109-111: The authors refer here to a study by Chen et al., 2015, which shows that galectin regulates susceptibility to H7N9 infection. According to the authors, this observation suggests an involvement of SPINK6 in protection from virus infection. However the link between galectin and SPINK6 remains unclear. This link has to be explained.
A: We cited the paper (Chen, Zhou et al. 2015), in which the original GWAS was described and the susceptibility gene galectin was characterized. Based on further data mining in the original GWAS and integrative analysis with eQTL datasets, we formulated the hypothesis of SPINK6 and conducted this study. Hence, galectin is nothing to do with SPINK6. However, after considering the comments of the other reviewers, we found this part of data is actually dispensable for formulating the hypothesis, instead it seems to cause a biased interpretation. We decide to change the data presentation and present them at the end of results.
A: We have corrected the mistake.
Referee #3 (Remarks for Author): The study by Wang et al. describes a previously reported inhibitor of serine protease families, namely SPINK6. The authors show that SPINK6 inhibits proteolytic cleavage of influenza HA and inhibits viral gene expression. The authors further explored the therapeutic potential of SPINK6 in mice. While some observations are interesting, the study is at a very premature stage. Following major concerns dampen my enthusiasm: 1. Database analysis reveal according to the authors a potential role for SPINK6 SNPs in human H7N9 infection by increased transcription. This is a potential important findings that needs to be experimentally validated. Data shown in Figure 1 are in silico data. The authors should use human biopsy material and assess SPINK6 expression in relation to key proteases, such as HAT or TMPRSS2 in the upper and lower respiratory tract. Also, they should study whether viral infection affects SPINK6 mRNA expression that could have major implications on the infection course.
A: We appreciate the reviewer's constructive comments. In brief, we have conducted a genome-wide genetic association study in 2013. Through integrative analysis of the genetic association results and eQTL datasets, we found that the risk variants to H7N9 infection (association data) are correlated to the higher SPINK6 expression level in human lung tissues (eQTL data). In the previous manuscript, this part of data was presented as one of evidences to formulate the hypothesis. After considering the reviewer's comment, we realize the presentation of this part of the data is not satisfactory. Hence, we re-organized the data presentation. In the revised manuscript, the genetic association and eQTL are presented at the end of the results as a supportive evidence in humans. This is actually the most common way of data presentation in many similar studies. We hope the revised manuscript would present our findings more rationally and explicitly.
The experiments suggested by the reviewer are very important. However, human biopsy materials are not readily available for research purpose; this is the reason why many eQTL datasets are generated and given access to all researchers. Moreover, it is difficult to quantitatively assess SPINK6 expression in relation to key proteases, especially the dynamic interplay of SPINK6 and these proteases, in human tissues, since it is quite challenging to maintain the viability of human tissues during such an experiment. We are the team establishing the first airway organoid model (Zhou, Li et al. 2018). In these physiologicallyactive airway organoids, we demonstrate that influenza virus infection upregulates SPINK6; the addition of SPINK6 protein or antibody significantly modulates the activities of endogenous proteases, HA cleavage and viral growth (the current Figure 4 or previous Figure  3). we hope the organoid data could adequately address the reviewer's inquiry.
2. There are no data showing that SPINK6 actually inhibits H7N9 replication. In Figure 2E only vRNA amounts are shown. The authors should show infectious virus titres as p.f.u. over several time points post infection.
A: We demonstrated SPINK6 suppression of trypsin-driven H7N9 replication by vRNA results ( Figure 2E). Additional data with infectious virus titer was also presented. SPINK6 suppressed HAT-activated H7N9 replication by plaque assay in the previous figure 4A (current Figure 3A). We always use multiple assays to reach a conclusion, including this study.
3. Animal data shown in Figure 6 are not allowing any conclusions. The authors have used a virus infectious dose of 5 p.f.u., which is too low to allow reproducible animal infection yet any meaningful conclusions. It is not clear why the authors used H1N1 influenza instead of H7N9 that is the primary focus of the study. It was repeatedly shown that H7N9 is able to infect mice and cause weight loss. The authors have used only 4 mice per time point assessed in Figure 6c. This is statistically not sufficient to allow significant conclusions. Further, the animal protocol is poorly described in the M&M section. There is no description of the narcosis used for infection and treatment. Humane endpoints are not defined.
A: We appreciate the reviewer's comment, from which we recognized an insufficient description of mouse experiments in the previous manuscript. A more detailed description of the mouse experiments and justification of experimental design are provided in the revised manuscript on page 9 and page 17. In this study, we have used a mouse-adapted strain of H1N1pdm virus. We demonstrated previously that robust viral growth of the virus in lung tissues led to a fatal outcome in young female Balb/c mice (Zheng, Chan et al. 2010). Survival rate is normally the golden standard to demonstrate the effect of an intervention in similar mouse studies. To compare the survival rate, we have allotted 10 mice per group for wildtype and mutant SPINK6 treatment. The significantly higher survival rate in wildtype-SPINK6-treated mice than mutant-SPINK6-treated mice (80% versus 30%) lends strong support to our hypothesis that SPINK6 inhibits the activation and propagation of IAVs. For detection of viral load and viral titer in lung tissues, we had 4 mice in each group, which may not be a big sample size. Nonetheless, survival rate may provide a more comprehensive evaluation of mouse infection. Importantly, the survival rate data is very consistent to viral growth.
We agree with the reviewer that a virus inoculation of 5 pfu is quite low in most cases. However, we'd like to direct the reviewer's attention to the design of the mouse experiments. Apart from virus inoculation, we performed 3 times of intranasal administration of SPINK6 proteins. The multiple intranasal administration itself exacerbated the infection and promoted viral growth even after a low MOI inoculation. Nevertheless, we observed a significantly lower lung virus titer in mice treated with wildtype SPINK6 than those treated with the mutant protein. 6 In mouse experiments, we used a mouse-adapted strain of H1N1pdm virus (Zheng, Chan et al. 2010). First, H1N1 viruses can be handled in P2 animal lab, which is less demanding than handling H7N9 virus in P3 animal lab. Secondly, we aimed to verify the in vitro findings, i.e., SPINK6 inhibition of virus activation and growth driven by serine proteases. Two subtypes of virus used as the targets of serine proteases, H1N1 and H7N9, share similar proteases for HA cleavage. Both can be cleaved by HAT ( Figure 2B), a major serine protease in the airway epithelium. As such, it doesn't matter which virus is used for the mouse experiment. We selected a mouse-adapted H1N1 strain, which is more readily handled in P2 lab. We'd like to emphasize that the primary focus of the study is SPINK6 inhibition of HA-activating proteases HAT and KLK5 rather than H7N9. These serine proteases can activate most influenza viruses including H1N1 and H7N9.
A: We thank the reviewer's comment for the description of mouse experiment, including euthanasia and the humane endpoint. We have amended the relevant part in materials and methods accordingly. 2nd Editorial Decision 18th May 2021 Dear Dr. Zhou, Thank you for the submission of your manuscript to EMBO Molecular Medicine. We have now heard back from the two referees who agreed to re-evaluate your manuscript. As you will see from the reports below, while the referee #1 is supporting publication of the study, referee #2 (previously #3) evaluated the revision as unsatisfactory particularly regarding your responses to the points #1 and #3. From the editorial side, we find you addressed the point #1 adequately, however, we agree with the referee #2 that the animal experiments are inconclusive.
Taking this in consideration it is clear that publication of the paper cannot be considered at this stage. I also note that addressing the reviewers concerns in full will be necessary for further considering the manuscript in our journal and this appears to require a lot of additional work and experimentation. I am unsure whether you will be able or willing to address those and return a revised manuscript within the six months deadline. On the other hand, given the potential interest of the findings, I would be willing to consider a revised manuscript with the understanding that the referee #2 (previously #3) concerns regarding animal experiments must be experimentally addressed and that acceptance of the manuscript would entail a second round of review.
Please note that EMBO Molecular Medicine encourages a single round of revision only and therefore, acceptance or rejection of the manuscript will depend on the completeness of your responses included in the next, final version of the manuscript. For this reason, and to save you from any frustrations in the end, I would strongly advise against returning an incomplete revision and would also understand your decision if you chose to rather seek rapid publication elsewhere at this stage.
I look forward to receiving your revised manuscript.
Should you find that the requested revisions are not feasible within the constraints outlined here and choose, therefore, to submit your paper elsewhere, we would welcome a message to this effect. The authors of the EMBO Molecular Medicine manuscript "SPINK6 inhibits human airway serine proteases and restricts influenza virus activation", present their findings related to the impact of proteases and anti-proteases on influenza hemagglutinin cleavage and infection. They focus on the serine protease inhibitor Kazal-type 6 (SPINK6), and the authors evaluate HA cleavage from HA0 into HA2 in the presence of known proteases, using SPINK6 as an anti-protease. They draw the conclusion that SPINK6 inhibits HAT and KLK5 protease activity, which restricts HA cleavage and is associated with reduced infectivity in human organoid and murine infection models. They then present human data that correlates genetic variation in SPINK 6 with susceptibility to H7N9 infection. Their overall conclusion is that SPINK6 is an inhibitor of serine proteases in a manner that restricts influenza A virus infection in the human respiratory tract. These findings are interesting, and the data are presented in a manner that tells an interesting story that highlights the impact of their findings in the field. General Comments: 1. This manuscript presents some interesting information that advances the field of viral pathogenesis. I have no major revisions to suggest.
Referee #2 (Comments on Novelty/Model System for Author): experiments used are not solid. thus, no conclusions can be drawn that support the authors ´ hypothesis.
Referee #2 (Remarks for Author): Unfortunately, the authors did not make a serious attempt to adress my major concerns. Particularly, regarding comment #1 and #3. Findings regarding comment#1 remain mostly in silico with no attempt to verify findings (at least some of them) experimentally. The most critical point however is comment#3. It is at this stage not possible to drwa meaningful conclusions from the animal experiment. A dose of pfu 5 is claimed to be lethal for 2009 pH1N1. This is very unusual. The authors should show LD50 data. Using only 4 animals per group is too low sample size not allowing robust conclusion. Humane endpoints are not defined, which are critical.

Point-to-point response
Review 2's comment. Unfortunately, the authors did not make a serious attempt to adress my major concerns. Particularly, regarding comment #1 and #3. Findings regarding comment#1 remain mostly in silico with no attempt to verify findings (at least some of them) experimentally. The most critical point however is comment#3. It is at this stage not possible to draw meaningful conclusions from the animal experiment. A dose of pfu 5 is claimed to be lethal for 2009 pH1N1. This is very unusual. The authors should show LD50 data. Using only 4 animals per group is too low sample size not allowing robust conclusion. Humane endpoints are not defined, which are critical.
A: We thank the reviewer's comments. we are introducing the mouse experiment in more detail in the revised manuscript on page 17. In this mouse model, the LD50 of the mouse-adapted H1N1 virus is 150 pfu based on our previous study (Zheng, Chan et al., 2010). As we have mentioned in the previous response letter, 3-time intranasal inoculations of SPINK6 protein solutions befor e and after the virus inoculation substantially exacerbated the infection. It has been documented that a much lower pfu inoculation should be done if the inoculated mice are intranasally administrated for intervention (Smee, von Itzstein et al., 2012). We agree with the reviewer on the issue of the sample size of the mouse experiment. We have repeated the mouse experiment with more mice and two time-points. The new data is presented in Figure 5C. In the previous revision, we have specified the humane endpoint, which may have been missed by the reviewer. We highlighted it on page 17. Thank you for the submission of your manuscript to EMBO Molecular Medicine. I am pleased to inform you that we will be able to accept your manuscript pending the following final amendments: 1) In the main manuscript file, please do the following: -Correct/answer the track changes suggested by our data editors by working from the attached document.
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3) For more information: There is space at the end of each article to list relevant web links for further consultation by our readers. Could you identify some relevant ones and provide such information as well? Some examples are patient associations, relevant databases, OMIM/proteins/genes links, author's websites, etc... 4) As part of the EMBO Publications transparent editorial process initiative (see our Editorial at http://embomolmed.embopress.org/content/2/9/329), EMBO Molecular Medicine will publish online a Review Process File (RPF) to accompany accepted manuscripts. This file will be published in conjunction with your paper and will include the anonymous referee reports, your point-by-point response and all pertinent correspondence relating to the manuscript. Let us know whether you agree with the publication of the RPF and as here, if you want to remove or not any figures from it prior to publication. Please note that the Authors checklist will be published at the end of the RPF. The additional animal experiments performed have now strengthened the conclusions drawn. Figure 5 is  Do the data meet the assumptions of the tests (e.g., normal distribution)? Describe any methods used to assess it.
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